An Integrated Strategy Reveals Complex Glycosylation of Erythropoietin Using Mass Spectrometry
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An Integrated Strategy Reveals Complex Glycosylation of Erythropoietin Using Mass Spectrometry. / Guan, Yudong; Zhang, Min; Gaikwad, Manasi; Voss, Hannah; Fazel, Ramin; Ansari, Samira; Shen, Huali; Wang, Jigang; Schlüter, Hartmut.
in: J PROTEOME RES, Jahrgang 20, Nr. 7, 02.07.2021, S. 3654-3663.Publikationen: SCORING: Beitrag in Fachzeitschrift/Zeitung › SCORING: Zeitschriftenaufsatz › Forschung › Begutachtung
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TY - JOUR
T1 - An Integrated Strategy Reveals Complex Glycosylation of Erythropoietin Using Mass Spectrometry
AU - Guan, Yudong
AU - Zhang, Min
AU - Gaikwad, Manasi
AU - Voss, Hannah
AU - Fazel, Ramin
AU - Ansari, Samira
AU - Shen, Huali
AU - Wang, Jigang
AU - Schlüter, Hartmut
PY - 2021/7/2
Y1 - 2021/7/2
N2 - The characterization of therapeutic glycoproteins is challenging due to the structural heterogeneity of the therapeutic protein glycosylation. This study presents an in-depth analytical strategy for glycosylation of first-generation erythropoietin (epoetin beta), including a developed mass spectrometric workflow for N-glycan analysis, bottom-up mass spectrometric methods for site-specific N-glycosylation, and a LC-MS approach for O-glycan identification. Permethylated N-glycans, peptides, and enriched glycopeptides of erythropoietin were analyzed by nanoLC-MS/MS, and de-N-glycosylated erythropoietin was measured by LC-MS, enabling the qualitative and quantitative analysis of glycosylation and different glycan modifications (e.g., phosphorylation and O-acetylation). The newly developed Python scripts enabled the identification of 140 N-glycan compositions (237 N-glycan structures) from erythropoietin, especially including 8 phosphorylated N-glycan species. The site-specificity of N-glycans was revealed at the glycopeptide level by pGlyco software using different proteases. In total, 114 N-glycan compositions were identified from glycopeptide analysis. Moreover, LC-MS analysis of de-N-glycosylated erythropoietin species identified two O-glycan compositions based on the mass shifts between non-O-glycosylated and O-glycosylated species. Finally, this integrated strategy was proved to realize the in-depth glycosylation analysis of a therapeutic glycoprotein to understand its pharmacological properties and improving the manufacturing processes.
AB - The characterization of therapeutic glycoproteins is challenging due to the structural heterogeneity of the therapeutic protein glycosylation. This study presents an in-depth analytical strategy for glycosylation of first-generation erythropoietin (epoetin beta), including a developed mass spectrometric workflow for N-glycan analysis, bottom-up mass spectrometric methods for site-specific N-glycosylation, and a LC-MS approach for O-glycan identification. Permethylated N-glycans, peptides, and enriched glycopeptides of erythropoietin were analyzed by nanoLC-MS/MS, and de-N-glycosylated erythropoietin was measured by LC-MS, enabling the qualitative and quantitative analysis of glycosylation and different glycan modifications (e.g., phosphorylation and O-acetylation). The newly developed Python scripts enabled the identification of 140 N-glycan compositions (237 N-glycan structures) from erythropoietin, especially including 8 phosphorylated N-glycan species. The site-specificity of N-glycans was revealed at the glycopeptide level by pGlyco software using different proteases. In total, 114 N-glycan compositions were identified from glycopeptide analysis. Moreover, LC-MS analysis of de-N-glycosylated erythropoietin species identified two O-glycan compositions based on the mass shifts between non-O-glycosylated and O-glycosylated species. Finally, this integrated strategy was proved to realize the in-depth glycosylation analysis of a therapeutic glycoprotein to understand its pharmacological properties and improving the manufacturing processes.
U2 - 10.1021/acs.jproteome.1c00221
DO - 10.1021/acs.jproteome.1c00221
M3 - SCORING: Journal article
C2 - 34110173
VL - 20
SP - 3654
EP - 3663
JO - J PROTEOME RES
JF - J PROTEOME RES
SN - 1535-3893
IS - 7
ER -